Low Molecular Weight Heparin Tinzaparin Research Articles

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans.

Circulating microRNAs (miRNAs) have become increasingly popular biomarker candidates in various diseases. However, heparin-based anticoagulants might affect the detection of target miRNAs in blood samples during quantitative polymerase chain reaction (qPCR)-based analysis of miRNAs involving RNA extraction, cDNA synthesis and the polymerase catalyzed reaction. Because low-molecular-weight heparins (LMWH) are widely used in routine healthcare, we aimed to investigate whether a prophylactic dose of the LMWH tinzaparin influences qPCR-based quantification of circulating miRNAs. A total of 30 subjects were included: 16 fracture patients with tinzaparin treatment and 14 non-fracture controls without anticoagulation therapy. To control for the effect of tinzaparin on miRNA analysis an identical concentration of synthetic miRNAs was added to plasma, isolated RNA and prepared complementary DNA (cDNA) from all samples in both groups. No significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin-treated patients with untreated controls. Among the tinzaparin-treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and one to six hours after a subcutaneous injection of tinzaparin 4500IU. No significant effect was observed for any of the investigated miRNAs. A prophylactic dose of 4500IU tinzaparin does not seem to affect cDNA synthesis or qRT-PCR-based quantification of circulating miRNAs.

Farmakomekanik katater aracılı tromboliz ile tedavi edilen akut ilyofemoral derin ven trombozlu hastalarda uzun süreli antikoagülasyonun karşılaştırmalı etkinliği

Aim: We aimed to compare the stability of pharmacologic profile, rate of symptomatic recurrent venous thromboembolism, major bleeding and the net clinical benefit on the regimen with vitamin K antagonist (VKA), low molecular weight heparin (LMWH) and direct oral anticoagulant (DOAC) for long-term anticoagulation in patients undergoing pharmaco-mechanical catheter-directed thrombolysis (PMCDT) for the treatment of deep vein thrombosis (DVT). Material and Methods: During the period from January 2019 until June 2019, data of 112 patients who underwent PMCDT for the treatment of acute iliofemoral DVT in our institution with long-term apixaban (Pfizer, Turkey) medication were prospectively collected (Group 1-DOAC). Data of control groups within January 2017- December 2018 period were collected retrospectively. Control groups consisted of PMCDT patients with extended LMWH (Tinzaparin, Abdi Ibrahim Pharma, Turkey) treatment (Group 2-LMWH; N=119) and with VKA (Coumadin, Eczacibasi Pharma, Turkey) treatment (Group 3- Control; N=111). Results: Patients treated with VKA showed a significant incompliance starting from third month up to one year. Patency rate diminished significantly below 70%. 32% of VKA patients were out of therapeutic range even in the first month leading to 40% at the end of the year. Likert Scale, Villalta/VCCS and VEINES-QOL-Sym scores confirmed the clinical data. Conclusion: This study highlights the potential role of DOAC as a reasonable alternative to VKAs/LMWH in the long-term anticoagulation strategy for DVT. We await larger clinical trials to support these findings and establish the role of DOAC as the standard of care for patients with DVT.

Impact of Anticoagulant and Antiplatelet on the Thrombotic Risk in Cancer Associated Thrombosis

Introduction: Therapeutic options for the management of venous thromboembolism (VTE) in patients with cancer remain very limited. Although low-molecular-weight heparin (LMWH) monotherapy has been identified as a simple and efficacious regimen. However, treatment of patients with a high risk of bleeding as well as the treatment of recurrent VTE with dose escalation of LMWH, and management in patients with thrombocytopenia represent major challenges. On the other side, Platelet activation is the first response to tissue damage and, may promote chronic inflammation-related cancer, mainly through the release of soluble factors and vesicles that are rich in genetic materials and proteins. Platelets also sustain cancer cell invasion and metastasis formation by fostering the development of the epithelial-mesenchymal transition phenotype, cancer cell survival in the bloodstream and arrest/extravasation at the endothelium. Furthermore, platelets contribute to tumor escape from immune elimination. Hence, we examined the effect of combined lose doses of the LMWH tinzaparin and the antiplatelet GPIIb/IIIa antagonist DMP728 on cancer associated thrombosis (CAT) using thrombelastogrphy. DMP728 is used for inhibiting the binding of Platelet GPIIb/IIIa. Glycoprotein IIb/IIIa is a receptor for fibrin and leads to further the activation of platelets to form blood clots. Aim: To determine the effects of tinzaparin and/or DMP728 on platelet/fibrin clot kinetics using thrombelastography (TEG) in presence or absence of pancreatic cancer cells (SUIT2) that expresses tissue factor. Methods: Human blood collected from healthy volunteers in sodium citrate tubes and treated with different concentrations (0.01, 0.03, 0.1 and 0.3 μg) of Tinzaparin, DMP 728, and/or tinzaparin + DMP728 in presence or absence of pancreatic cancer cells (SUIT 2 cells). We compared the effects on the coagulation time, time to clot initiation and thrombus strength level in vitro. Results: In the absence of SUIT 2 pancreatic cancer cells, thrombelastographic (TEG) have shown that, increasing concentrations of tinzaparin and DMP728 (0.01 - 0.3 μg) were associated with marked increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. After adding the SUIT 2 pancreatic cancer cells to human blood, shortened the time to clot initiation (R) and increased the clot strength (MA) have been observed. In presence of SUIT2 Pancreatic Cancer Cell, tinzaparin was associated with increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. However, DMP728 R Value has not shown much effect on clotting time (R) and maximum amplitude of the clot formation (MA). A combination of DMP728 and tinzaparin was associated with a greater synergistic Increase in clotting time (R), with a synergistic decrease in maximum amplitude of the clot formation (MA) (Figure 1). Conclusions: The combination of the LMWH (tinzaparin) and the antiplatelet drug (DMP728) might be a successful approach in the prevention and treatment of cancer associated thrombosis. Disclosures No relevant conflicts of interest to declare.

The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways.

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets’ ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

Heparin antagonizes cisplatin resistance of A2780 ovarian cancer cells by affecting the Wnt signaling pathway.

Low molecular weight heparin (LMWH), the guideline based drug for prophylaxis and treatment of cancer-associated thrombosis, was recently shown to sensitize cisplatin resistant A2780cis human ovarian cancer cells for cisplatin cytotoxicity upon 24 h pretreatment with 50 μg × mL−1 of the LMWH tinzaparin in vitro, equivalent to a therapeutic dosage. Thereby, LMWH induced sensitization by transcriptional reprogramming of A2780cis cells via not yet elucidated mechanisms that depend on cellular proteoglycans. Here we aim to illuminate the underlying molecular mechanisms of LMWH in sensitizing A2780cis cells for cisplatin. Using TCF/LEF luciferase promotor assay (Top/Flash) we show that resistant A2780cis cells possess a threefold higher Wnt signaling activity compared to A2780 cells. Furthermore, Wnt pathway blockade by FH535 leads to higher cisplatin sensitivity of A2780cis cells. Glypican-3 (GPC3) is upregulated in A2780cis cells in response to LMWH treatment, probably as counter-regulation to sustain the high Wnt activity against LMWH. Hence, LMWH reduces the cisplatin-induced rise in Wnt activity and TCF-4 expression in A2780cis cells, but keeps sensitive A2780 cells unaffected. Consequently, Wnt signaling pathway appears as primary target of LMWH in sensitizing A2780cis cells for cisplatin toxicity. Considering the outstanding role of LMWH in clinical oncology, this finding appears as promising therapeutic option to hamper chemoresistance.

The Impact of the Low Molecular Weight Heparin Tinzaparin on the Sensitization of Cisplatin-Resistant Ovarian Cancers-Preclinical In Vivo Evaluation in Xenograft Tumor Models.

Resistance formation of tumors against chemotherapeutics is the major obstacle in clinical cancer therapy. Although low molecular weight heparin (LMWH) is an important component in oncology referring to guideline-based antithrombotic prophylaxis of tumor patients, a potential interference of LMWH with chemoresistance is unknown. We have recently shown that LMWH reverses the cisplatin resistance of A2780cis human ovarian cancer cells in vitro. Here we address the question whether this LMWH effect is also valid under in vivo conditions. Therefore, we established tumor xenografts of A2780 and cisplatin resistant A2780cis cells in nude mice and investigated the impact of daily tinzaparin applications (10 mg/kg BW) on anti-tumor activity of cisplatin (6 mg/kg BW, weekly) considering the tumor growth kinetics. Intratumoral platinum accumulation was detected by GF-AAS. Xenografts of A2780 and A2780cis cells strongly differed in cisplatin sensitivity. As an overall consideration, tinzaparin co-treatment affected the response to cisplatin of A2780cis, but not A2780 tumors in the later experimental time range. A subgroup analysis confirmed that initially smaller A2780cis tumors benefit from tinzaparin, but also small A2780 xenografts. Tinzaparin did not affect cisplatin accumulation in A2780cis xenografts, but strongly increased the platinum content in A2780, obviously related to morphological differences in both xenografts. Although we cannot directly confirm a return of A2780cis cisplatin resistance by tinzaparin, as shown in vitro, the present findings give reason to discuss heparin effects on cytostatic drug efficiency for small tumors and warrants further investigation.

In Surgical Colon Cancer Patients Extended-Duration Thromboprophylaxis (30 days) with the Highest Dose of Tinzaparin (4,500 IU s.c./q.d.) Normalizes the Postoperative VEGF Levels.

Background/Purpose: In colon cancer (CC) patients preoperative (pre-op) levels of VEGF-A165 (VEGF) is a strong predictor for disease recurrence. Elevated postoperative (post-op) VEGF levels could have undesirable effects by enhancing tumor growth and metastasis formation. It has been suggested that thromboprophylaxis with a Low Molecular Weight Heparin (LMWH) in surgical cancer patients, further to thromboembolic protection, may exert some anti-neoplastic properties, as well. The aim of our study was to assess the potential impact of the LMWH Tinzaparin (Innohep® - Leo Pharma, Copenhagen, Denmark), given at different doses and for different perioperative (peri-op) periods, upon the post-op variability of serum VEGF levels in surgical CC patients.Methods: A total of 54 consecutive CC patients who underwent a curative resection were randomized in four groups according to their peri-op thromboprophylaxis scheme, which was based on administrating Tinzaparin in different doses and at different periods, as follows: group I: 3,500 IU for 10 days, group II: 3,500 IU for 30 days, group III: 4,500 IU for 10 days and group IV: 4,500 IU for 30 days. Serum VEGF concentrations were evaluated on the pre-op day (Day 0) and on the 10th and 30th post-op days (Day 10 and Day 30, respectively). For statistical analyses the mixed design ANOVA was used. P < 0.05 was considered significant.Results: On Day 0, VEGF didn't differ between groups I, II, III and IV (p>0.05, for every comparison). On Day 10, VEGF was increased in all groups. Between Day 10 and Day 30, VEGF remained stable in groups I (p=0.031) and II (p=1.000) and increased significantly in group III (p=0.005). On the contrary, VEGF decreased significantly in group IV (p<0.001). The most remarkable finding was observed when we compared VEGF between Day 0 and Day 30: while in groups I, II and III, VEGF remained significantly higher compared to Day 0 (p<0.001, p=0.041 and p<0.001, respectively), on the contrary, in group IV (extended-duration with the highest dose of 4,500 IU of tinzaparin) it was comparable to Day 0 (p=1.000).Conclusions: In surgical CC patients only the recommended thromboprophylaxis scheme with the highest prophylactic dose of Tinzaparin (4,500 IU) for extended-duration (30 days) normalizes VEGF levels at the end of the first post-op month by reducing them to the pre-op levels.

Endothelial Protective Properties of Low Molecular Weight Heparin (LMWH) Tinzaparin in Combination with Statin: A Novel Potential Therapeutic Tool in Preventing Cancer Metastasis?

Introduction8.2 million people die from cancer worldwide every year. LMWH prolongs survival in newly-diagnosed, limited stage lung cancer. Interestingly, trials recruiting patients with other cancers have also demonstrated improved survival in patients with long life-expectancy but not in advanced malignancy, suggesting that the benefit may be mediated by metastasis prevention. LMWHs inhibit metastasis in vivo but may cause bleeding complications when used at effective doses. Consequently, LMWH is unlikely to be used for metastasis prevention unless bleeding side-effects can be urgently addressed. Enhanced endothelial barrier (EB) permeability is a hallmark of metastasis and permits the extravasation of circulating tumour cells into the tissues of the target organ.AimsTo characterise LMWH Tinzaparin-mediated EB protection and underlying mechanisms and to optimise its cytoprotective properties in a manner that may reduce the associated bleeding risk.MethodsEA.hy926 endothelial monolayers were exposed to LMWH (including a novel LMWH formulation comprised of a fraction of LMWH tinzaparin consisting of short polysaccharide chains, mean MW 2.8KDa), simvastatin (a lipid-lowering agent shown to exhibit EB protective properties) and either metastasis-associated agonists (thrombin & VEGF) or calcein blue-AM labelled metastatic tumour cells (DU145, prostate carcinoma). EB function was assessed through the measurement of the migration of an Evans blue-conjugated albumin solution/labelled tumour cells through the monolayers. The role of PAR-1 signalling in modulating permeability was assessed by flow cytometry and permeability assays using an anti-PAR-1 cleavage site antibody and PAR-1 activating/inhibitory peptides. Endothelial myosin light chain-2 diphosphorylation status (MLC-2; the key determinant of endothelial cell actin cytoskeleton contraction) was determined by SDS-PAGE and western blotting. The anticoagulant activity of the LMWHs was determined by calibrated automated thrombography (CAT) and plasma anti-factor Xa activity assays.ResultsTinzaparin treatment prior to thrombin (IIa) and VEGF exposure significantly attenuated agonist-induced EB permeability in a concentration-dependent manner. This cytoprotective effect was observed within a clinically relevant concentration range (0-2IU/mL). Incubation of endothelial monolayers with LMWH also significantly reduced the migration of metastatic carcinoma cells (fluorescence of culture media containing labelled migrated tumour cells reduced to 73.9±5.7% of baseline; p<0.01).IIa-induced permeability was found to be entirely mediated through PAR-1 cleavage. IIa-induced permeability was lost in the presence of an anti-PAR-1 receptor antibody and a PAR-1 signalling inhibitory peptide. Tinzaparin did not alter EA.hy926 PAR-1 expression and did not inhibit IIa-mediated PAR-1 cleavage but reduced IIa-induced MLC-2 phosphorylation and attenuated PAR-1 activating peptide-induced permeability, suggesting that the protective effect of LMWH is not mediated through an inhibition of IIa proteolytic activity (and is therefore independent of its anticoagulant activity).The 2.8KDa LMWH fraction exhibited diminished anticoagulant activity relative to Tinzaparin but retained EB protective properties and attenuated tumour cell trans-migration (at a concentration equivalent to 0.5IU/mL, tumour cell migration was reduced to 76.4±4.7% of baseline and IIa-induced albumin permeability was attenuated to 66.9±8.9%).Remarkably, the cytoprotective properties of Tinzaparin were significantly enhanced when combined with a statin. The combination of a sub-anticoagulant concentration of Tinzaparin (0.1IU/mL) and a clinically relevant concentration of simvastatin (20nM), attenuated EB permeability to a significantly greater extent that that induced by each agent alone (IIa-induced EB permeability was reduced to 7.9±0.2% of baseline (p<0.05)).ConclusionLMWH enhances the barrier function of endothelium in vitro. This effect is not linked to its anticoagulant activity. Strategies which permit using either non-anticoagulant LMWH or sub-anticoagulant LMWH concentrations in combination with other barrier-protective agents may represent a means of delivering this potentially anti-metastatic activity of LMWH to patients without conferring a bleeding risk. DisclosuresMaguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion.

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS-RBCs) to stimulated cultured endothelial cells invitro. Importantly, sevuparin prevents vaso-occlusion and normalizes blood flow in an invivo mouse model of SCD vaso-occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P- and L-selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso-occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti-adhesive efficacy similar to the low molecular weight heparin tinzaparin both invitro and invivo. These results suggest that the anti-adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso-occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

The presence of heparins during decidualization modulates the response of human endometrial stromal cells to IL-1β in vitro.

The aim of this paper is to study the impact of heparin on the response of human endometrial stromal cells (ESCs) to interleukin (IL)-1β during decidualization in vitro. ESCs were isolated from hysterectomy specimens of premenopausal women undergoing hysterectomy for benign reasons; decidualized in vitro and incubated in parallel with unfractionated heparin or tinzaparin; and stimulated with IL-1β at days 0, 3, 6, and 9 during decidualization. IL-6, IL-11, and leukemia inhibitory factor (LIF) were analyzed using ELISAs and real-time RT-PCR. Cell viability was determined by a fluorometric assay. IL-1β dose-dependently stimulated IL-6, IL-11, and LIF in distinct patterns in ESCs during decidualization. Unfractionated heparin as well as tinzaparin attenuated the IL-1β-mediated induction of IL-6, IL-11, and LIF on protein and messenger RNA (mRNA) levels. The relative effects of heparin and tinzaparin were getting more pronounced during the time course of decidualization. Unfractionated heparin and the low molecular weight heparin tinzaparin have modulating effects on IL-1β-induced endometrial cytokines of the IL-6 family during decidualization. These effects of heparins beyond their classical anti-coagulatory properties might have implications on the regulation of endometrial receptivity and early implantation.

Low molecular heparin for cancer-associated venous thromboembolism – still the “CATCH of the day“?

SummaryIn December 2014 the CATCH study was presented at the annual meeting of the American Society of Hematology. CATCH is a randomized controlled trial comparing the low molecular weight heparin tinzaparin with warfarin for 6 months extended treatment (=secondary prophylaxis) of cancer-associated venous thromboembolism (VTE). 6.9 % of patients in the tanzaparin arm experienced recurrent symptomatic and asymptomatic VTE compared with 10 % in the warfarin arm, this difference was statistically not significant. The difference became significant when only symptomatic VTEs were compared. There was no difference in the incidence of major bleeding events, but significantly fewer patients experienced clinically relevant non-major bleeding with tinzaparin than warfarin. The CATCH study is so far the largest study on extended treatment of cancer-associated VTE. It supports the guideline recommendation that low molecular weight heparins should be preferred to vitamin K antagonists for anticoagulation of cancer-associated VTE.

Low molecular weight heparin tinzaparin antagonizes cisplatin resistance of ovarian cancer cells

Low molecular weight heparin (LMWH) is routinely used for antithrombotic treatment of cancer patients. Preclinical- and clinical data suggest that LMWH has beneficial effects for cancer patients beyond the prevention of thrombosis, i.e. by inhibiting metastasis. It is, however, unclear whether heparin has an impact on the efficiency of chemotherapy in cancer patients. Here we show that a therapeutic dosage of LMWH tinzaparin reverses cisplatin resistance of A2780cis human ovarian cancer cells to the level of sensitive cells. This novel activity of tinzaparin is associated with intense transcriptional reprogramming. Our gene expression profiling experiments revealed that 3776 genes responded to tinzaparin treatment. For this reason tinzaparin has a complex impact on diverse biological processes. We discovered that tinzaparin inhibits the expression of genes that mediate cisplatin resistance of A2780cis cells. In contrast tinzaparin induced the expression of genes that antagonize drug resistance. This activity of tinzaparin is mediated by cell surface proteoglycans, since enzymatic cleavage of heparan sulfates prevented the reversal of cisplatin resistance. These data indicate that cell surface heparan sulfate proteoglycans play an important role for chemotherapy resistance. The results of this study shed a new light on LMWH application in cancer therapy and suggest tinzaparin as promising treatment option of ovarian cancer patients in combination with anticancer drugs. Future clinical trials are needed to validate these findings.

Investigation of the antiangiogenic behaviors of rivaroxaban and low molecular weight heparins

Antithrombotic agents play important roles in the prophylactic and therapeutic management of many cardiovascular disorders. Therefore, many researchers have focused on developing new strategies for anticoagulation. New oral anticoagulants and factor Xa inhibitors are products of such research. Although they are identified as advantageous, there are limited data available about their multisystemic interactions. Thus, the antiangiogenic behaviors of oral factor Xa inhibitors and low molecular weight heparins (LMWHs) were investigated in this study. The chick chorioallantoic membrane (CAM) model was designed to investigate the antiangiogenic potential of new oral factor Xa inhibitors (rivaroxaban) and LMWHs (enoxaparin sodium and tinzaparin sodium). Four different molar concentrations (10, 10, 10, and 10 μmol/l) were studied for each drug. Each concentration was studied on 20 fertilized eggs. Vessel structures were evaluated under a stereoscopic microscope, and vessel formation was scaled according to previous literature. Both enoxaparin and tinzaparin sodium have increased antiangiogenic efficacy on CAM in a dose-dependent manner. However, this increased efficacy did not reach significant levels (average score < 0.5). On the contrary, while rivaroxaban showed dose-dependent antiangiogenic properties similar to enoxaparin and tinzaparin, a significant average antiangiogenic score (0.7) was detected at 10 μmol/l concentrations. New oral anticoagulants seem to be more favorable. However, their safety for the cardiovascular system needs to be clarified through microsystem studies on, for example, angiogenesis.

Suppression of pancreatic cancer by sulfated non-anticoagulant low molecular weight heparin

Sulfated non-anticoagulant heparins (S-NACHs) might be preferred for potential clinical use in cancer patients without affecting hemostasis as compared to low molecular weight heparins (LMWHs). We investigated anti-tumor effects, anti-angiogenesis effects, and mechanisms of S-NACH in a mouse model of pancreatic cancer as compared to the LMWH tinzaparin. S-NACH or tinzaparin with or without gemcitabine were administered, and tumor luminescent signal intensity, tumor weight, and histopathology were assessed at the termination of the study. S-NACH and LMWH efficiently inhibited tumor growth and metastasis, without any observed bleeding events with S-NACH as compared to tinzaparin. S-NACH distinctly increased tumor necrosis and enhanced gemcitabine response in the mouse pancreatic cancer models. These data suggest the potential implication of S-NACH as a neoadjuvant in pancreatic cancer.

Skin necrosis associated with thromboprophylaxis after total knee replacement.

Thromboprophylaxis are routinely given to prevent venous thromboembolism (VTE) in patients after total hip and knee replacement surgeries. Low molecular weight heparin (LMWH) (fractioned heparin) is effective in the prevention and treatment of VTE. The predicable effect of LMWH has popularized it for routine clinical use. Although LMWH has lesser complication rate, compared to unfractioned heparin (UFH), sporadic clinical complication has been reported. We report a rare case of skin necrosis secondary to use of LMWH tinzaparin used for routine thromboprophylaxis after total knee replacement.

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

CATCH: a randomised clinical trial comparing long-term tinzaparin versus warfarin for treatment of acute venous thromboembolism in cancer patients

BackgroundLow-molecular-weight heparin (LMWH) is recommended and commonly used for extended treatment of cancer-associated thrombosis (CAT), but its superiority over warfarin has been demonstrated in only one randomised study. We report here the rationale, design and a priori analysis plans of Comparison of Acute Treatments in Cancer Haemostasis (CATCH; NCT01130025), a multinational, Phase III, open-label, randomised controlled trial comparing tinzaparin with warfarin for extended treatment of CAT.Methods/DesignThe primary objective is to assess the efficacy of tinzaparin in preventing recurrent venous thromboembolism (VTE) in patients with active cancer and acute, symptomatic proximal deep vein thrombosis and/or pulmonary embolism. The secondary objectives are to determine: safety of tinzaparin given over 6 months; clinical and laboratory markers for recurrent VTE and/or major bleeding; 6-month overall mortality; incidence and severity of post-thrombotic syndrome; patient-reported quality of life; and healthcare resource utilisation. Nine hundred patients are randomised to receive tinzaparin 175 IU/kg once daily for 6 months or initial tinzaparin 175 IU/kg once daily for 5–10 days and dose-adjusted warfarin (target INR 2.0–3.0) for 6 months. The primary composite outcome is time to recurrent VTE, including incidental VTE and fatal pulmonary embolism. All patients are followed up to 6 months or death, whichever comes sooner. Blinded adjudication will be performed for all reported VTE, bleeding events and causes of death. Efficacy will be analysed using centrally adjudicated results of all patients according to intention-to-treat analysis. An independent Data Safety Monitoring Board is reviewing data at regular intervals and an interim analysis is planned after 450 patients have completed the study.DiscussionThe results will add significantly to the knowledge of the efficacy, safety and cost effectiveness of tinzaparin in the prevention of recurrent VTE in patients with cancer and thrombosis. Prospective data will emerge on the clinical significance of incidental VTE and risk stratification in patients with CAT. Results on post-thrombotic syndrome, quality of life and healthcare resource utilisation will inform decision makers on how to secure better patient care. If tinzaparin is shown to be more effective than warfarin, CATCH will provide valuable confirmatory data to support the use of the LMWH tinzaparin for extended treatment of CAT.

Are low molecular weight heparins able to sensitize chemoresistant tumor cells?

Venous thromboembolic complications are often associated with malignant diseases. The treatment and prevention of these events in cancer patients is frequently carried out using low-molecular-weight heparin (LMWH). However, heparin has other pharmacological effects in addition to the antithrombotic activities [1], e.g., antimetastatic and antiangiogenetic activities and these are currently subjects of intensive research [2]. In clinical trials with cancer patients it has been shown that treatment with LMWH is associated with an increase in survival rate and this effect of LMWH is not only attributable to its prevention of lethal thromboembolic complications [3]. Recent additional findings show that unfractioned heparin (UFH) can alter the chemoresistance of tumor cells by interference with P-glycoprotein (P-gp)-mediated multidrug resistance [4]. Therefore, it appears worthwhile to investigate whether LMWH acts as a potential synergist or modulator of chemotherapy. However, published data on this topic are rare. The aim of this in vitro study was to obtain an insight into the possible interaction between LMWH and P-gp in multidrug resistant cells and to investigate whether this can be considered as a plausible mechanism explaining the improvement in survival of cancer patients receiving anticoagulant treatment. In this study the influence of the LMWH tinzaparin on Pgp-independent chemoresistance phenomenon such as that associated with cisplatin resistance was also investigated.

Inhibitory effect of non-anticoagulant heparin (S-NACH) on pancreatic cancer cell adhesion and metastasis in human umbilical cord vessel segment and in mouse model

Metastasis is the most devastating aspect of cancer and it is the main cause of morbidity and mortality in cancer patients. Tumor cell adhesion to the vascular endothelial cell lining is an important step in metastatic progression and is prompted by platelets. Mucin 1 is over-expressed and aberrantly glycosylated in more than 60% of pancreatic ductal adeno-carcinomas, which mediate adhesion of pancreatic cancer cells to platelets via P-selectin. The anticoagulant low molecular weight heparins (LMWHs), which are commonly used in venous Thromboprophylaxis and treatment, appear to have an effect on cancer survival. The aim of this study is to investigate the effect of platelets on human pancreatic cancer MPanc96 cell adhesion to the endothelial cell vessel wall, and to examine the effect of heparin derivatives on MPanc96 adhesion using a novel, in vitro model of human umbilical cord vein. The modified heparin S-NACH (sulfated non-anticoagulant heparin), which is devoid of antithrombin (AT) binding and devoid of inhibition of systemic AT-dependent coagulation factors such as factor Xa and IIa, and the LMWH tinzaparin both potently reduced adhesion and invasion of fluorescence-labeled MPanc96 cancer cells to the endothelial layer of umbilical cord vein in a dose-dependent manner. S-NACH effectively inhibited P-selectin mediated MPanc96 cell adhesion, and inhibited cell adhesion and invasion similar to tinzaparin, indicating that systemic anticoagulation is not a necessary component for heparin attenuation of cancer cell adhesion, invasion, and metastasis. Also, S-NACH and tinzaparin versus unfractionated heparin, heparin derivatives enoxaparin, deltaparin, fraxiparin, and fondaparinux were evaluated for their effect on platelet-cancer cell adhesion. An in vivo anti-metastatic S-NACH-treated nude mouse model of MPanc96 pancreatic cancer cell metastasis demonstrated potent anti-metastasis efficacy as evidenced by IVIS imaging and histological staining.

Comparative Pharmacodynamic Assessment of the Antiangiogenesis Activity of Heparin and Low-Molecular-Weight Heparin Fractions

Effects of unfractionated heparin and low-molecular-weight heparins (LMWHs) on human microvascular endothelial cell sprouting (tube formation assay) in vitro were determined. Antiangiogenesis efficacy of commercially available LMWHs tinzaparin and enoxaparin in the chick chorioallantoic membrane (CAM) model of growth factor-induced angiogenesis was compared. The LMWH tinzaparin was fractionated into different molecular weight (MW) pools by size exclusion chromatography; they inhibited CAM angiogenesis depending on their MW distribution, with optimal inhibition at 8 to 12 kDa and no inhibition at <2 kDa. Tinzaparin demonstrated greater antiangiogenesis efficacy than enoxaparin (P < .001); these CAM results correlated with the endothelial tube formation assay results (P < .001, tinzaparin vs enoxaparin). These data point to the variable antiangiogenesis efficacy of different LMWHs as a function of MW and perhaps other structural differences. Our hypothesis confirmed a relationship between lower release of tissue factor pathway inhibitor by lower MW fractions of tinzaparin or enoxaparin leading to reduced antiangiogenesis efficacy.